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Screening and identification of DnaJ interaction proteins in Streptococcus pneumoniae.

Cai Y, Yan W, Xu W, Yin Y, He Y, Wang H, Zhang X - Curr. Microbiol. (2013)

Bottom Line: The resulting protein mixtures were subjected to SDS-PAGE and those differential bands were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry.Furthermore, to confirm the interaction between DnaJ and these candidates, bacterial two-hybrid system was employed to reveal, for example, the interaction between DnaJ and DnaK, Eno, SpxB.Further protein expression experiments suggested that DnaJ prevented denaturation of Eno and SpxB at high temperature.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, China.

ABSTRACT
Streptococcus pneumoniae DnaJ is recognized as a virulence factor whose role in pneumococcal virulence remains unclear. Here, we attempted to reveal the contribution of DnaJ in pneumococcal virulence from the identification of its interacting proteins using co-immunoprecipitation method. dnaJ was cloned into plasmid pAE03 generating pAE03-dnaJ-gfp which was used to transform S. pneumoniae D39 strain. Then anti-GFP coated beads were used to capture GFP-coupled proteins from the bacterial lysate. The resulting protein mixtures were subjected to SDS-PAGE and those differential bands were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. We finally obtained nine proteins such as DnaK, Gap, Eno, SpxB using this method. Furthermore, to confirm the interaction between DnaJ and these candidates, bacterial two-hybrid system was employed to reveal, for example, the interaction between DnaJ and DnaK, Eno, SpxB. Further protein expression experiments suggested that DnaJ prevented denaturation of Eno and SpxB at high temperature. These results help to understand the role of DnaJ in the pathogenesis of S. pneumoniae.

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DnaJ prevents the denaturation of Eno and SpxB during heat shock. S. pneumoniae D39, D39ΔdnaJ, D39-pEVP3-eno/spxB, and D39ΔdnaJ-pEVP3-eno/spxB were cultured in C+Y medium and all the bacteria incubated in 42 °C water bath for 0, 2, 5, 10, 20, 40, and 60 min separately. DnaJ/SpxB/Eno expressions in the bacterial lysates of D39 and D39ΔdnaJ were analyzed by western blot separately. CodY served as the internal reference (a). Figure b and c were the ratios of DnaJ/Eno versus CodY and DnaJ/SpxB versus CodY. The results of β-galactosidase reporter gene assay were indicated by Fig. d, e, and the figures represented three independent experiments. Statistical differences were analyzed by two-way ANOVA between groups of D39-pEVP3-eno/spxB and D39, D39ΔdnaJ or D39ΔdnaJ-pEVP3-eno/spxB, respectively. Their P values were marked with *** located on the right of the spots. For D39-pEVP3-eno/spxB and D39-pEVP3-eno supernatant, statistical differences were compared by Student’s t test between samples at different time of heat shock and the initial time. Their P values were indicated above the spots. * indicates P < 0.05; ** indicates P < 0.01, and *** indicates P < 0.001
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Fig4: DnaJ prevents the denaturation of Eno and SpxB during heat shock. S. pneumoniae D39, D39ΔdnaJ, D39-pEVP3-eno/spxB, and D39ΔdnaJ-pEVP3-eno/spxB were cultured in C+Y medium and all the bacteria incubated in 42 °C water bath for 0, 2, 5, 10, 20, 40, and 60 min separately. DnaJ/SpxB/Eno expressions in the bacterial lysates of D39 and D39ΔdnaJ were analyzed by western blot separately. CodY served as the internal reference (a). Figure b and c were the ratios of DnaJ/Eno versus CodY and DnaJ/SpxB versus CodY. The results of β-galactosidase reporter gene assay were indicated by Fig. d, e, and the figures represented three independent experiments. Statistical differences were analyzed by two-way ANOVA between groups of D39-pEVP3-eno/spxB and D39, D39ΔdnaJ or D39ΔdnaJ-pEVP3-eno/spxB, respectively. Their P values were marked with *** located on the right of the spots. For D39-pEVP3-eno/spxB and D39-pEVP3-eno supernatant, statistical differences were compared by Student’s t test between samples at different time of heat shock and the initial time. Their P values were indicated above the spots. * indicates P < 0.05; ** indicates P < 0.01, and *** indicates P < 0.001

Mentions: DnaJ is helpful to the correct functions of protein, we supposed whether the interacting partners were regulated by DnaJ in this way. Because the correct function of protein is necessary for the activity of β-galactosidase, β-galactosidase reporter assay was used to reflect the portion of functional proteins in the bacterial lysates and the supernatants, while western blot was employed to determine the total synthesized proteins in different bacterial lysates. We have known that DnaJ can facilitate some secretory proteins transport across the membrane, such as alkaline phosphatase (AP), ribose-binding protein (RBP), and β-lactamase (Bla) and metallo-β-lactamase (MβL) in E. coli [26, 36, 37]. And it has been reported that enolase can be secreted outside the bacteria [2], so we were also interested in the level of Eno in the supernatant. The amount of functional Eno was much more in wild type D39 strain than D39ΔdnaJ as revealed by β-galactosidase activity analysis (Fig. 4d), and the total synthesized Eno was also elevated with the increasing expression of DnaJ (Fig. 4a, b). Besides, we observed that the secretion of Eno was in parallel with the expression of DnaJ (Fig. 4b, d). It was suggested that besides its effect on the prevention of denaturation of Eno, DnaJ might also help to promote its outside secretion.Fig. 4


Screening and identification of DnaJ interaction proteins in Streptococcus pneumoniae.

Cai Y, Yan W, Xu W, Yin Y, He Y, Wang H, Zhang X - Curr. Microbiol. (2013)

DnaJ prevents the denaturation of Eno and SpxB during heat shock. S. pneumoniae D39, D39ΔdnaJ, D39-pEVP3-eno/spxB, and D39ΔdnaJ-pEVP3-eno/spxB were cultured in C+Y medium and all the bacteria incubated in 42 °C water bath for 0, 2, 5, 10, 20, 40, and 60 min separately. DnaJ/SpxB/Eno expressions in the bacterial lysates of D39 and D39ΔdnaJ were analyzed by western blot separately. CodY served as the internal reference (a). Figure b and c were the ratios of DnaJ/Eno versus CodY and DnaJ/SpxB versus CodY. The results of β-galactosidase reporter gene assay were indicated by Fig. d, e, and the figures represented three independent experiments. Statistical differences were analyzed by two-way ANOVA between groups of D39-pEVP3-eno/spxB and D39, D39ΔdnaJ or D39ΔdnaJ-pEVP3-eno/spxB, respectively. Their P values were marked with *** located on the right of the spots. For D39-pEVP3-eno/spxB and D39-pEVP3-eno supernatant, statistical differences were compared by Student’s t test between samples at different time of heat shock and the initial time. Their P values were indicated above the spots. * indicates P < 0.05; ** indicates P < 0.01, and *** indicates P < 0.001
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Related In: Results  -  Collection

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Fig4: DnaJ prevents the denaturation of Eno and SpxB during heat shock. S. pneumoniae D39, D39ΔdnaJ, D39-pEVP3-eno/spxB, and D39ΔdnaJ-pEVP3-eno/spxB were cultured in C+Y medium and all the bacteria incubated in 42 °C water bath for 0, 2, 5, 10, 20, 40, and 60 min separately. DnaJ/SpxB/Eno expressions in the bacterial lysates of D39 and D39ΔdnaJ were analyzed by western blot separately. CodY served as the internal reference (a). Figure b and c were the ratios of DnaJ/Eno versus CodY and DnaJ/SpxB versus CodY. The results of β-galactosidase reporter gene assay were indicated by Fig. d, e, and the figures represented three independent experiments. Statistical differences were analyzed by two-way ANOVA between groups of D39-pEVP3-eno/spxB and D39, D39ΔdnaJ or D39ΔdnaJ-pEVP3-eno/spxB, respectively. Their P values were marked with *** located on the right of the spots. For D39-pEVP3-eno/spxB and D39-pEVP3-eno supernatant, statistical differences were compared by Student’s t test between samples at different time of heat shock and the initial time. Their P values were indicated above the spots. * indicates P < 0.05; ** indicates P < 0.01, and *** indicates P < 0.001
Mentions: DnaJ is helpful to the correct functions of protein, we supposed whether the interacting partners were regulated by DnaJ in this way. Because the correct function of protein is necessary for the activity of β-galactosidase, β-galactosidase reporter assay was used to reflect the portion of functional proteins in the bacterial lysates and the supernatants, while western blot was employed to determine the total synthesized proteins in different bacterial lysates. We have known that DnaJ can facilitate some secretory proteins transport across the membrane, such as alkaline phosphatase (AP), ribose-binding protein (RBP), and β-lactamase (Bla) and metallo-β-lactamase (MβL) in E. coli [26, 36, 37]. And it has been reported that enolase can be secreted outside the bacteria [2], so we were also interested in the level of Eno in the supernatant. The amount of functional Eno was much more in wild type D39 strain than D39ΔdnaJ as revealed by β-galactosidase activity analysis (Fig. 4d), and the total synthesized Eno was also elevated with the increasing expression of DnaJ (Fig. 4a, b). Besides, we observed that the secretion of Eno was in parallel with the expression of DnaJ (Fig. 4b, d). It was suggested that besides its effect on the prevention of denaturation of Eno, DnaJ might also help to promote its outside secretion.Fig. 4

Bottom Line: The resulting protein mixtures were subjected to SDS-PAGE and those differential bands were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry.Furthermore, to confirm the interaction between DnaJ and these candidates, bacterial two-hybrid system was employed to reveal, for example, the interaction between DnaJ and DnaK, Eno, SpxB.Further protein expression experiments suggested that DnaJ prevented denaturation of Eno and SpxB at high temperature.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, China.

ABSTRACT
Streptococcus pneumoniae DnaJ is recognized as a virulence factor whose role in pneumococcal virulence remains unclear. Here, we attempted to reveal the contribution of DnaJ in pneumococcal virulence from the identification of its interacting proteins using co-immunoprecipitation method. dnaJ was cloned into plasmid pAE03 generating pAE03-dnaJ-gfp which was used to transform S. pneumoniae D39 strain. Then anti-GFP coated beads were used to capture GFP-coupled proteins from the bacterial lysate. The resulting protein mixtures were subjected to SDS-PAGE and those differential bands were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. We finally obtained nine proteins such as DnaK, Gap, Eno, SpxB using this method. Furthermore, to confirm the interaction between DnaJ and these candidates, bacterial two-hybrid system was employed to reveal, for example, the interaction between DnaJ and DnaK, Eno, SpxB. Further protein expression experiments suggested that DnaJ prevented denaturation of Eno and SpxB at high temperature. These results help to understand the role of DnaJ in the pathogenesis of S. pneumoniae.

Show MeSH
Related in: MedlinePlus